1. Field of the Invention
This invention relates generally to the technique of fluorescence microscopy which has developed recently into a valuable tool for biological research. Fluorescence microscopy is an extraordinary sensitive method for detecting low concentrations of a substance which comprises treating the substance with a dye to produce a complex which fluoresces when excited with incident light. More particularly, this disclosure relates to a method of detecting qualitatively and/or quantitatively a variety of organisms in biological samples. The detection of said organisms is facilitated by the nucleic acid staining properties of the disclosed dyes.
2. Description of the Prior Art
A variety of compounds have been used in fluorescent microscopy histology and fluorescence microfluorimetry to selectively stain nucleic acids. Von Bertalanffy and Bickis, J. Histochem., Cytochem., Vol. 4, pp. 481-493 (1956), reported that acridine orange could be used for the identification of cytoplasmic basophilia (RNA) by fluorescence microscopy. In particular, they found that acridine orange enables identification of basophilic cytoplasmic inclusions in the supravital state, and that the red fluorescent cytoplasmic inclusions correspond to those stained with the toluidine blue technique and shown by ribonuclease to consist mainly of RNA.
Rosell, et al reported in Nature, Vol. 253, p. 461, (1975) that 4'6-diamidini-2-phenylindole (DAPI) has been shown to possess useful DNA binding properties. Specifically, they found that DAPI can be used as a highly specific fluorescent stain for both nuclear and mitochondrial DNA in yeast.
Hilwig and Gropp in Experimental Cell Research, 75, pp. 122-126 (1972) discuss a simple and direct fluorescence staining procedure using a benzimidazole derivative (identified as 33258 Hoechst) to visualize chromosomal segments to heterchromatin and (in the mouse) sites of repetitious DNA.
However, there are various disadvantages associated with the prior art dyes that are employed in fluorescent microscopy and related techniques. The complexes produced by conventionally employed dyes have limited excitation/emission spectra, low quantum yields, high background fluorescence, low fluorescent enhancement and most importantly such dye complexes are not permanent but rather tend to fade and bleach. In addition, most of the prior art dyes have a high degree of fluorescence in the free, uncomplexed state and are relatively unstable when exposed to light. For example, complexes produced as a result of treating DNA with acridine orange exhibit a fluorescent enhancement only twice as great as acridine orange in the free uncomplexed state. In addition, DAPI and Hoechst 33258, although producing complexes having detectable fluorescent enhancements when bound to DNA, require ultraviolet excitation and emit a blue fluorescence thereby increasing the difficulty of observing such complexes using fluorescent microscopy. There exists a need in fluorescent microscopy and related techniques for a class of dyes essentially nonfluorescent in the free, uncomplexed state and capable of forming highly fluorescent complexes when bound to cellular components, in particular, nucleic acids. It is further desired that such complexes have low background fluorescence, a high degree of fluorescent enhancement and be preferably metachromic and permanent.
Some of the compounds utilized in the method of the present invention have been described as laser dyes in U.S. Pat. No. 3,833,863 and as photographic dyes in Defensive Publication T88 9016. However, neither reference discloses nor suggests the use of these compounds as proposed in the present invention.